LOCUS       sequence-364437-        6783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd gen lentiviral backbone for cloning and expression of new shRNA 
            sequences. Uses blasticidin for selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6783)
  AUTHORS   Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, 
            Martin-Belmonte F, Mostov KE
  TITLE     A molecular network for de novo generation of the apical surface and
            lumen.
  JOURNAL   Nat Cell Biol. 2010 Nov;12(11):1035-45. doi: 10.1038/ncb2106. Epub 
            2010 Oct 3.
  PUBMED    20890297
REFERENCE   2  (bases 1 to 6783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/ncb2106"; 
            journalName: "Nat Cell Biol"; date: "2010-11"; volume: "12"; issue: 
            "11"; pages: "1035-45"
COMMENT     Sequence Label: pLKO.1-blast
FEATURES             Location/Qualifiers
     source          1..6783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(164..752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(244..263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(923..1714,1715..1783))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1546..1565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1784..1888)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1914..2369)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2051..2072)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2263..2282
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2496..2518
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2510..2527
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2511..2527
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2537..2556
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2537..2555
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2576..2711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2751..2872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2762..2781)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2816..2835
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2944..3177
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(3249..3647)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(3659..4169)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3722..3740)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    4218..4335
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(4398..4646)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4456..4475)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4626..4646)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(4944..4988)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5173..5406
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5899..6024
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6071..6251
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6252..6478)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(6506..6526)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6506..6524)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6545..6561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6545..6561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6558..6580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6569..6585
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6593..6599,6600..6617,6618..6623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6638..6659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(6776..6783,1..10))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
       61 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
      781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
      841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
      901 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
      961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1021 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1081 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1201 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     1261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1741 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1861 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     1921 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     1981 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2041 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2101 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2161 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2221 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2281 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2341 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     2401 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2461 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2521 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtacgt ccctaggcct
     2581 ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct
     2641 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     2701 aggggcggga tgggcggagt taggggcggg atagctagag ccagacatga taagatacat
     2761 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2821 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttcctctcac
     2881 tctctgatat tcatttcttt gcaagttata aatactgaat aataagatga catgaactac
     2941 tactgctaga gattttccac actgactaaa agggtctgag ggatctctag ttaccagagt
     3001 cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat tgaggcttaa
     3061 gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct aaccagagag
     3121 acccagtaca agcaaaaagc agatcttgtc ttcgttggga gtgaattagc ccttccagtc
     3181 cccccttttc ttttaaaaag tggctaagat ctacagctgc cttgtaagtc attggtctta
     3241 aaggtacctt agccctccca cacataacca gagggcagca attcacgaat cccaactgcc
     3301 gtcggctgtc catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc
     3361 acctgtcggc accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg
     3421 atacaagtca ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca
     3481 caaggtcccc cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag
     3541 agagctgcgc tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt
     3601 gctctttcaa tgagggtgga ttcttcttga gacaaaggct tggccatggt ggatccccct
     3661 ggggagagag gtcggtgatt cggtcaacga gggagccgac tgccgacgtg cgctccggag
     3721 gcttgcagaa tgcggaacac cgcgcgggca ggaacagggc ccacactacc gccccacacc
     3781 ccgcctcccg caccgcccct tcccggccgc tgctctcggc gcgccctgct gagcagccgc
     3841 tattggccac agcccatcgc ggtcggcgcg ctgccattgc tccctggcgc tgtccgtctg
     3901 cgagggtact agtgagacgt gcggcttccg tttgtcacgt ccggcacgcc gcgaaccgca
     3961 aggaaccttc ccgacttagg ggcggagcag gaagcgtcgc cggggggccc acaagggtag
     4021 cggcgaagat ccgggtgacg ctgcgaacgg acgtgaagaa tgtgcgagac ccagggtcgg
     4081 cgccgctgcg tttcccggaa ccacgcccag agcagccgcg tccctgcgca aacccagggc
     4141 tgccttggaa aaggcgcaac cccaaccccc tcgagccgcg gccaaagtgg atctctgctg
     4201 tccctgtaat aaacccgaaa attttgaatt tttgtaattt gtttttgtaa ttctttagtt
     4261 tgtatgtctg ttgctattat gtctactatt ctttcccctg cactgtaccc cccaatcccc
     4321 ccttttcttt taaaattgtg gatgaatact gccatttgtc tcgaggtcga gaattcacgc
     4381 gtgcatacct gcggaccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg
     4441 aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact
     4501 gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg
     4561 tttacagtca aattaattcc aattatctct ctaacagcct tgtatcgtat atgcaaatat
     4621 gaaggaatca tgggaaatag gccctcggtg aagggggcgg ccgctcgagg ctagtctcgt
     4681 gatcgatacc gtcgagatcc gttcactaat cgaatggatc tgtctctgtc tctctctcca
     4741 ccttcttctt ctattccttc gggcctgtcg ggtcccctcg gggttgggag gtgggtctga
     4801 aacgataatg gtgaatatcc ctgcctaact ctattcacta tagaaagtac agcaaaaact
     4861 attcttaaac ctaccaagcc tcctactatc attatgaata attttatata ccacagccaa
     4921 tttgttatgt taaaccaatt ccacaaactt gcccatttat ctaattccaa taattcttgt
     4981 tcattctttt cttgctggtt ttgcgattct tcaattaagg agtgtattaa gcttgtgtaa
     5041 ttgttaattt ctctgtccca ctccatccag gtcgtgtgat tccaaatctg ttccagagat
     5101 ttattactcc aactagcatt ccaaggcaca gcagtggtgc aaatgagttt tccagagcaa
     5161 ccccaaatcc ccaggagctg ttgatccttt aggtatcttt ccacagccag gattcttgcc
     5221 tggagctgct tgatgcccca gactgtgagt tgcaacagat gctgttgcgc ctcaatagcc
     5281 ctcagcaaat tgttctgctg ctgcactata ccagacaata attgtctggc ctgtaccgtc
     5341 agcgtcattg acgctgcgcc catagtgctt cctgctgctc ccaagaaccc aaggaacaaa
     5401 gctcctattc ccactgctct tttttctctc tgcaccactc ttctctttgc cttggtgggt
     5461 gctactccta atggttcaat ttttactact ttatatttat ataattcact tctccaattg
     5521 tccctcatat ctcctcctcc aggtctgaag atcagcggcc gcttgctgtg cggtggtctt
     5581 acttttgttt tgctcttcct ctatcttgtc taaagcttcc ttggtgtctt ttatctctat
     5641 cctttgatgc acacaataga gggttgctac tgtattatat aatgatctaa gttcttctga
     5701 tcctgtctga agggatggtt gtagctgtcc cagtatttgt ctacagcctt ctgatgtttc
     5761 taacaggcca ggattaactg cgaatcgttc tagctccctg cttgcccata ctatatgttt
     5821 taatttatat tttttctttc cccctggcct taaccgaatt ttttcccatc gcgatctaat
     5881 tctcccccgc ttaatactga cgctctcgca cccatctctc tccttctagc ctccgctagt
     5941 caaaattttt ggcgtactca ccagtcgccg cccctcgcct cttgccgtgc gcgcttcagc
     6001 aagccgagtc ctgcgtcgag agagctcctc tggtttccct ttcgctttca agtccctgtt
     6061 cgggcgccac tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta
     6121 ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga
     6181 ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac
     6241 cagagagacc cgtttatgta tcgagctagg cacttaaata caatatctct gcaatgcggc
     6301 aattcagtgg ttcgtccaat ccatgtcaga cccgtctgtt gccttcctaa taaggcacga
     6361 tcgtaccacc ttacttccac caatcggcat gcacggtgct ttttctctcc ttgtaaggca
     6421 tgttgctaac tcatcgttac catgttgcaa gactacaaga gtattgcata agactacatt
     6481 aagcttgcag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     6541 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     6601 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6661 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6721 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6781 tca
//
